Adeno associated virus vectors for the treatment of a cardiomyopathy

ABSTRACT

The use of recombinant adeno-associated virus (AAV) virions for delivery of DNA molecules to muscle cells and tissue is disclosed. The invention allows for the direct, in vivo injection of recombinant AAV virions into muscle tissue, e.g., by intramuscular injection, as well as for the in vitro transduction of muscle cells which can subsequently be introduced into a subject for treatment. The invention provides for sustained, high-level expression of the delivered gene and for in vivo secretion of the therapeutic protein from transduced muscle cells such that systemic delivery is achieved.

This application is a continuation of U.S. patent application Ser. No.09/406,362, filed Sep. 28, 1999 U.S. Pat. No. 6,335,011, which is acontinuation of U.S. patent application Ser. No. 08/784,757, filed Jan.16, 1997 U.S. Pat. No. 5,962,313, which is a continuation-in-part ofU.S. patent application Ser. No. 08/588,355, filed Jan. 18, 1996 U.S.Pat. No. 5,858,351, from which applications priority is claimed pursuantto 35 U.S.C. §120, and which applications are incorporated herein byreference in their entireties.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation-in-part of U.S. application Ser. No.08/588,355, filed Jan. 18, 1996, from which priority is claimed pursuantto 35 USC §120 and which is incorporated herein by reference in itsentirety.

TECHNICAL FIELD

The present invention relates generally to DNA delivery methods. Moreparticularly, the invention relates to the use of recombinantadeno-associated virus (AAV) virions for delivery of a selected gene tomuscle cells and tissue. The method provides for sustained, high-levelexpression of the delivered gene.

BACKGROUND OF THE INVENTION

Gene delivery is a promising method for the treatment of acquired andinherited diseases. Muscle tissue is an appealing gene delivery targetbecause it is readily accessible, well-differentiated and nondividing.Barr and Leiden (1991) Science 254:1507-1509. These properties areimportant in the selection of appropriate delivery strategies to achievemaximal gene transfer.

Several experimenters have demonstrated the ability to deliver genes tomuscle cells with the subsequent systemic circulation of proteinsencoded by the delivered genes. See, e.g., Wolff et al. (1990) Science247:1465-1468; Acsadi et al. (1991) Nature 352:815-818; Barr and Leiden(1991) Science 254:1507-1509; Dhawan et al. (1991) Science254:1509-1512; Wolff et al. (1992) Human Mol. Genet. 1:363-369; Eyal etal. (1993) Proc. Nat. Acad. Sci. USA 90:4523-4527; Davis et al. (1993)Hum. Gene Therapy 4:151-159.

Genes have been delivered to muscle by direct injection of plasmid DNA,such as described by Wolff et al. (1990) Science 247:1465-1468; Acsadiet al. (1991) Nature 352:815-818; Barr and Leiden (1991) Science254:1507-1509. However, this mode of administration generally results insustained but low levels of expression. Low but sustained expressionlevels may be effective in certain situations, such as for providingimmunity.

Viral based systems have also been used for gene delivery to muscle. Forexample, human adenoviruses are double-stranded DNA viruses which entercells by receptor-mediated endocytosis. These viruses have beenconsidered well suited for gene transfer because they are easy to growand manipulate and they exhibit a broad host range in vivo and in vitro.Adenoviruses are able to infect quiescent as well as replicating targetcells and persist extrachromosomally, rather than integrating into thehost genome.

Despite these advantages, adenovirus vectors suffer from severaldrawbacks which make them ineffective for long term gene therapy. Inparticular, adenovirus vectors express viral proteins that may elicit animmune response which may decrease the life of the transduced cell. Thisimmune reaction may preclude subsequent treatments because of humoraland/or T cell responses. Furthermore, the adult muscle cell may lack thereceptor which recognizes adenovirus vectors, precluding efficienttransduction of this cell type using such vectors. Thus, attempts to useadenoviral vectors for the delivery of genes to muscle cells hasresulted in poor and/or transitory expression. See, e.g., Quantin et al.(1992) Proc. Natl. Acad. Sci. USA 89:2581-2584; Acsadi et al. (1994)Hum. Mol. Genetics 3:579-584; Acsadi et al. (1994) Gene Therapy1:338-340; Dai et al. (1995) Proc. Natl. Acad. Sci. USA 92:1401-1405;Descamps et al. (1995) Gene Therapy 2:411-417; Gilgenkrantz et al.(1995) Hum. Gene Therapy 6:1265-1274.

Gene therapy methods based upon surgical transplantation of myoblastshas also been attempted. See, e.g., International Publication no. WO95/13376; Dhawan et al. (1991) Science 254:1509-1512; Wolff et al.(1992) Human Mol. Genet. 1:363-369; Dai et al. (1992) Proc. Natl. Acad.Sci. USA 89:10892-10895; Hamamori et al. (1994) Hum. Gene Therapy5:1349-1356; Hamamori et al. (1995) J. Clin. Invest. 95:1808-1813; Blauand Springer (1995) New Eng. J. Med. 333:1204-1207; Leiden, J. M. (1995)New Eng. J. Med. 333:871-872; Mendell et al. (1995) New Eng. J. Med.333:832-838; and Blau and Springer (1995) New Eng. J. Med.333:1554-1556. However, such methods require substantial tissue culturemanipulation and surgical expertise, and, at best, show inconclusiveefficacy in clinical trials. Thus, a simple and effective method of genedelivery to muscle, resulting in long-term expression of the deliveredgene, would be desirable.

Recombinant vectors derived from an adeno-associated virus (AAV) havebeen used for gene delivery. AAV is a helper-dependent DNA parvoviruswhich belongs to the genus Dependovirus. AAV requires infection with anunrelated helper virus, such as adenovirus, a herpesvirus or vaccinia,in order for a productive infection to occur. The helper virus suppliesaccessory functions that are necessary for most steps in AAVreplication. In the absence of such infection, AAV establishes a latentstate by insertion of its genome into a host cell chromosome. Subsequentinfection by a helper virus rescues the integrated copy which can thenreplicate to produce infectious viral progeny. AAV has a wide host rangeand is able to replicate in cells from any species so long as there isalso a successful infection of such cells with a suitable helper virus.Thus, for example, human AAV will replicate in canine cells coinfectedwith a canine adenovirus. AAV has not been associated with any human oranimal disease and does not appear to alter the biological properties ofthe host cell upon integration. For a review of AAV, see, e.g., Bernsand Bohenzky (1987) Advances in Virus Research (Academic Press, Inc.)32:243-307.

The AAV genome is composed of a linear, single-stranded DNA moleculewhich contains approximately 4681 bases (Berns and Bohenzky, supra). Thegenome includes inverted terminal repeats (ITRs) at each end whichfunction in cis as origins of DNA replication and as packaging signalsfor the virus. The internal nonrepeated portion of the genome includestwo large open reading frames, known as the AAV rep and cap regions,respectively. These regions code for the viral proteins involved inreplication and packaging of the virion. For a detailed description ofthe AAV genome, see, e.g., Muzyczka, N. (1992) Current Topics inMicrobiol. and Immunol. 158:97-129.

The construction of recombinant AAV (rAAV) virions has been described.See, e.g., U.S. Pat. Nos. 5,173,414 and 5,139,941; InternationalPublication Numbers WO 92/01070 (published Jan. 23, 1992) and WO93/03769 (published Mar. 4, 1993); Lebkowski et al. (1988) Molec. Cell.Biol. 8:3988-3996; Vincent et al. (1990) Vaccines 90 (Cold Spring HarborLaboratory Press); Carter, B. J. (1992) Current Opinion in Biotechnology3:533-539; Muzyczka, N. (1992) Current Topics in Microbiol. and Immunol.158:97-129; and Kotin, R. M. (1994) Human Gene Therapy 5:793-801.

Recombinant AAV virion production generally involves cotransfection of aproducer cell with an AAV vector plasmid and a helper construct whichprovides AAV helper functions to complement functions missing from theAAV vector plasmid. In this manner, the producer cell is capable ofexpressing the AAV proteins necessary for AAV replication and packaging.The AAV vector plasmid will include the DNA of interest flanked by AAVITRs which provide for AAV replication and packaging functions. AAVhelper functions can be provided via an AAV helper plasmid that includesthe AAV rep and/or cap coding regions but which lacks the AAV ITRs.Accordingly, the helper plasmid can neither replicate nor packageitself. The producer cell is then infected with a helper virus toprovide accessory functions, or with a vector which includes thenecessary accessory functions. The helper virus transactivates the AAVpromoters present on the helper plasmid that direct the transcriptionand translation of AAV rep and cap regions. Upon subsequent culture ofthe producer cells, recombinant AAV virions harboring the DNA ofinterest, are produced.

Recombinant AAV virions have been shown to exhibit tropism forrespiratory epithelial cells (Flotte et al. (1992) Am. J. Respir. CellMol. Biol. 7:349-356; Flotte et al. (1993) J. Biol. Chem. 268:3781-3790;Flotte et al. (1993) Proc. Natl. Acad. Sci. USA 90:10613-10617) andneurons of the central nervous system (Kaplitt et al. (1994) NatureGenetics 8:148-154). These cell types are well-differentiated,slowly-dividing or postmitotic. Flotte et al. (1993) Proc. Natl. Acad.Sci. USA 90:10613-10617; Kaplitt et al. (1994) Nature Genetics8:148-154. The ability of AAV vectors to transduce nonproliferatingcells (Podsakoff et al. (1994) J. Virol. 68:5656-5666; Russell et al.(1994) Proc. Natl. Acad. Sci. USA 91:8915-8919; Flotte et al. (1994) Am.J. Respir. Cell Mol. Biol. 11:517-521) along with the attributes ofbeing inherently defective and nonpathogenic, place AAV in a uniqueposition among gene therapy viral vectors.

Despite these advantages, the use of recombinant AAV virions to delivergenes to muscle cells in vivo has not heretofore been disclosed.

SUMMARY OF THE INVENTION

Accordingly, the present invention is based on the surprising andunexpected discovery that recombinant AAV (rAAV) virions provide forefficient delivery of genes and sustained production of therapeuticproteins in various muscle cell types. The invention allows for in vivosecretion of the therapeutic protein from transduced muscle cells suchthat systemic delivery of therapeutic levels of the protein is achieved.These results are seen with both in vivo and in vitro modes of DNAdelivery. Hence, rAAV virions allow delivery of DNA directly to muscletissue. The ability to deliver and express genes in muscle cells, aswell as to provide for secretion of the produced protein from transducedcells, allows the use of gene therapy approaches to treat and/or preventa wide variety of disorders.

Furthermore, the ability to deliver DNA to muscle cells by intramuscularadministration in vivo provides a more efficient and convenient methodof gene transfer.

Thus, in one embodiment, the invention relates to a method of deliveringa selected gene to a muscle cell or tissue. The method comprises:

(a) providing a recombinant AAV virion which comprises an AAV vector,the AAV vector comprising the selected gene operably linked to controlelements capable of directing the in vivo transcription and translationof the selected gene; and

(b) introducing the recombinant AAV virion into the muscle cell ortissue.

In particularly preferred embodiments, the selected gene encodes atherapeutic protein, such as erythropoietin (EPO), or the lysosomalenzyme, acid α-glucosodase (GAA).

In another embodiment, the invention is directed to a muscle cell ortissue transduced with a recombinant AAV virion which comprises an AAVvector, the AAV vector comprising a selected gene operably linked tocontrol elements capable of directing the in vivo transcription andtranslation of the selected gene.

In still further embodiments, the invention is directed to a method oftreating an acquired or inherited disease in a mammalian subjectcomprising introducing into a muscle cell or tissue of the subject, invivo, a therapeutically effective amount of a pharmaceutical compositionwhich comprises (a) a pharmaceutically acceptable excipient; and (b)recombinant AAV virions. The recombinant AAV virions comprise an AAVvector, the AAV vector comprising a selected gene operably linked tocontrol elements capable of directing the transcription and translationof the selected gene when present in the subject.

In yet another embodiment, the invention is directed to a method oftreating an acquired or inherited disease in a mammalian subjectcomprising:

(a) introducing a recombinant AAV virion into a muscle cell or tissue invitro to produce a transduced muscle cell. The recombinant AAV virioncomprises an AAV vector, the AAV vector comprising a selected geneoperably linked to control elements capable of directing thetranscription and translation of the selected gene when present in thesubject; and

(b) administering to the subject a therapeutically effective amount of acomposition comprising a pharmaceutically acceptable excipient and thetransduced muscle cells from step (a).

In a further embodiment, the invention relates to a method fordelivering a therapeutically effective amount of a protein systemicallyto a mammalian subject comprising introducing into a muscle cell ortissue of the subject a pharmaceutical composition which comprises (a) apharmaceutically acceptable excipient; and (b) recombinant AAV virions,wherein the recombinant AAV virions comprise an AAV vector, the AAVvector comprising a selected gene operably linked to control elementscapable of directing the transcription and translation of the selectedgene when present in the subject, wherein the introducing is done invivo.

In another embodiment, the invention is directed to a method fordelivering a therapeutically effective amount of a protein systemicallyto a mammalian subject comprising:

(a) introducing a recombinant AAV virion into a muscle cell or tissue invitro to produce a transduced muscle cell, wherein the recombinant AAVvirion comprises an AAV vector, the AAV vector comprising a selectedgene operably linked to control elements capable of directing thetranscription and translation of the selected gene when present in thesubject; and

(b) administering to the subject a therapeutically effective amount of acomposition comprising a pharmaceutically acceptable excipient and thetransduced muscle cells from step (a).

In other embodiments, the invention is directed to an AAV vectorcomprising a gene encoding either erythropoietin (EPO), or acidα-glucosidase (GAA), operably linked to control elements capable ofdirecting the in vivo transcription and translation of the gene, as wellas a recombinant AAV (rAAV) virion comprising the vector.

These and other embodiments of the subject invention will readily occurto those of ordinary skill in the art in view of the disclosure herein.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows in situ histochemical detection of β-galactosidaseexpression in murine muscle cells following transduction with rAAV-LacZas described in Example 3, Part A. In the study, the tibialis anteriormuscle of adult Balb/c mice was injected with 8×10⁹ rAAV-LacZ. Animalswere sacrificed (a) 2, (b) 4, (c) 8, (d) 12, (e) 24, of (f) 32 weeksafter injection. The tibialis anterior was excised, and 10 mm sectionswere stained by X-gal for β-galactosidase histochemistry. The stainedtissue samples were photographed at 25×.

FIG. 2 shows a section of skeletal muscle two months after injectionwith rAAV-LacZ as described in Example 3, Part A. The tibialis anteriormuscle was processed for in situ detection of β-galactosidaseexpression, and photographed with diffraction-interference contrastoptics at 400×.

FIG. 3 depicts β-galactosidase expression in Balb/c mice tibialisanterior muscle transduced in vivo with rAAV-LacZ as described inExample 3, Part B. Adult Balb/c mice were injected intramuscularly (IM)with various doses of rAAV-LacZ. At 2 and 8 weeks post injection, tissuewas harvested for analysis of beta-galactosidase (β-gal). β-galexpression was analyzed by measurement of relative light units (RLU)emitted from muscle homogenates, as detected by luminometer.

FIG. 4 shows the secretion of human erythropoietin (hEPO) fromtransduced myotubes and myoblasts, as described in Example 4. Myotubes(differentiated cells) or myoblasts (actively dividing cells) weretransduced with rAAV-hEPO at a ratio of approximately 10⁵ per targetcell. Levels of secreted hEPO were analyzed in supernatants at varioustime points. Baseline levels of hEPO prior to transduction were belowthe level of detection in both cell populations; the values at each timepoint represent replicate values+/−standard deviation.

FIG. 5 shows the secretion of human erythropoietin (hEPO) by C2C12myotubes transduced with rAAV-hEPO as described in Example 4. ConfluentC2C12 myoblasts were differentiated into myotubes and transduced with3×10⁸ (open bar), 3×10⁹ (cross-hatched bar), or 3×10¹⁰ (solid bar)rAAV-hEPO. Secretion of EPO was measured 3, 8, and 14 days aftertransduction. Control rAAV-LacZ myotubes secreted <2.5 mU/mL EPO. Thebar graph depicts mean production of EPO/well/24 hour as determined intriplicate cultures±the standard error of mean (SEM).

FIG. 6 shows the secretion of human erythropoietin (hEPO) by primaryhuman myotubes transduced with rAAV-hEPO as described in Example 5.Confluent human myoblasts were differentiated into myotubes by culturefor 14 days in reduced-serum media, then transduced with 3×10⁸ (openbar), 3×10⁹ (cross-hatched bar), or 3×10¹⁰ (solid bar) rAAV-hEPO.Secretion of EPO was measured 3, 8 and 14 days after transduction.Control myotubes transduced with rAAV-LacZ secreted <2.5 mU/mL EPO. Thebar graph depicts mean production of EPO/well/24 hour as determined intriplicate cultures±SEM.

FIG. 7 depicts the time course of EPO secretion in Balb/c mice after IMinjection with rAAV-hEPO. Adult Balb/c mice were injected IM with 1×10¹⁰(▾), 3×10¹⁰ (▴) 1×10¹¹ (▪), or 3×10¹¹ () purified rAAV-LacZ at day=0,and serum EPO levels measured at various time points after injection.Reported values represent means (n=4)±SEM.

FIG. 8 shows high level expression of acid α-glucsidase (GAA) in humanskeletal muscle transduced in vitro with rAAV-hGAA as described inExample 8, Part A. In the study, differentiated human myoblasts wereexposed to rAAV-hGAA virions at a MOI of 2×10⁵. Cells were collected atthe time points indicated, and GAA activity measured by enzymatic assay.Non-transduced control cells (open bar) and cells transduced withrAAV-LacZ (cross-hatched bar) showed no significant expression of GAA,while cells transduced with rAAV-hGAA (solid bar) showed high levels ofGAA activity. The bar graph represents mean GAA activity determined intriplicate cultures±SEM.

FIG. 9 shows expression of acid α-glucosidase in Balb/c mice tibialisanterior muscle cells that were transduced in vivo with rAAV-hGAA, asdescribed in Example 8, Part B. Adult Balb/c mice were injectedintramuscularly (IM) with 4×10¹⁰ rAAV-hGAA (solid bar) or the same doseof rAAV-LacZ (open bar). At various time points after injection, muscletissue was harvested for analysis of GAA activity by enzymatic assay.The bar graph shows mean GAA activity determined in five animals (weeks1 and 4), or in four animals (week 10)±SEM.

DETAILED DESCRIPTION OF THE INVENTION

The practice of the present invention will employ, unless otherwiseindicated, conventional methods of virology, microbiology, molecularbiology and recombinant DNA techniques within the skill of the art. Suchtechniques are explained fully in the literature. See, e.g., Sambrook,et al. Molecular Cloning: A Laboratory Manual (Current Edition); DNACloning: A Practical Approach, vol. I & II (D. Glover, ed.);Oligonucleotide Synthesis (N. Gait, ed., Current Edition); Nucleic AcidHybridization (B. Hames & S. Higgins, eds., Current Edition);Transcription and Translation (B. Hames & S. Higgins, eds., CurrentEdition); CRC Handbook of Parvoviruses, vol. I & II (P. Tijssen, ed.);Fundamental Virology, 2nd Edition, vol. I & II (B. N. Fields and D. M.Knipe, eds.)

All publications, patents and patent applications cited herein, whethersupra or infra, are hereby incorporated by reference in their entirety.

As used in this specification and the appended claims, the singularforms “a,” “an” and “the” include plural references unless the contentclearly dictates otherwise.

A. Definitions

In describing the present invention, the following terms will beemployed, and are intended to be defined as indicated below.

The phrase “gene delivery” or “gene transfer” refers to methods orsystems for reliably inserting foreign DNA into target cells, such asinto muscle cells. Such methods can result in transient or long termexpression of genes. Gene transfer provides a unique approach for thetreatment of acquired and inherited diseases. A number of systems havebeen developed for gene transfer into mammalian cells. See, e.g., U.S.Pat. No. 5,399,346.

The term “therapeutic protein” refers to a protein which is defective ormissing from the subject in question, thus resulting in a disease stateor disorder in the subject, or to a protein which confers a benefit tothe subject in question, such as an antiviral, antibacterial orantitumor function. A therapeutic protein can also be one which modifiesany one of a wide variety of biological functions, such as endocrine,immunological and metabolic functions. Representative therapeuticproteins are discussed more fully below.

By “vector” is meant any genetic element, such as a plasmid, phage,transposon, cosmid, chromosome, virus, virion, etc., which is capable ofreplication when associated with the proper control elements and whichcan transfer gene sequences between cells. Thus, the term includescloning and expression vehicles, as well as viral vectors.

By “AAV vector” is meant a vector derived from an adeno-associated virusserotype, including without limitation, AAV-1, AAV-2, AAV-3, AAV-4,AAV-5, AAVX7, etc. AAV vectors can have one or more of the AAV wild-typegenes deleted in whole or part, preferably the rep and/or cap genes(described below), but retain functional flanking ITR sequences (alsodescribed below). Functional ITR sequences are necessary for the rescue,replication and packaging of the AAV virion. Thus, an AAV vector isdefined herein to include at least those sequences required in cis forreplication and packaging (e.g., functional ITRs) of the virus. The ITRsneed not be the wild-type nucleotide sequences, and may be altered,e.g., by the insertion, deletion or substitution of nucleotides, so longas the sequences provide for functional rescue, replication andpackaging.

By “recombinant virus” is meant a virus that has been geneticallyaltered, e.g., by the addition or insertion of a heterologous nucleicacid construct into the particle.

By “AAV virion” is meant a wild-type (wt) AAV virus particle (comprisinga linear, single-stranded AAV nucleic acid genome associated with an AAVcapsid protein coat). In this regard, single-stranded AAV nucleic acidmolecules of either complementary sense, e.g., “sense” or “antisense”strands, can be packaged into any one AAV virion and both strands areequally infectious.

A “recombinant AAV virion,” or “rAAV virion” is defined herein as aninfectious, replication-defective virus composed of an AAV proteinshell, encapsidating a DNA molecule of interest which is flanked on bothsides by AAV ITRs. An rAAV virion is produced in a suitable producercell which has had an AAV vector, AAV helper functions and accessoryfunctions introduced therein. In this manner, the producer cell isrendered capable of encoding AAV polypeptides that are required forpackaging the AAV vector (containing a recombinant nucleotide sequenceof interest) into recombinant virion particles for subsequent genedelivery.

The term “transfection” is used to refer to the uptake of foreign DNA bya mammalian cell. A cell has been “transfected” when exogenous DNA hasbeen introduced inside the cell membrane. A number of transfectiontechniques are known in the art. See, e.g., Graham et al. (1973)Virology, 52:456, Sambrook et al. (1989) Molecular Cloning, a laboratorymanual, Cold Spring Harbor Laboratories, New York, Davis et al. (1986)Basic Methods in Molecular Biology, Elsevier, and Chu et al. (1981) Gene13:197. Such techniques can be used to introduce one or more exogenousDNA moieties, such as a plasmid vector and other nucleic acid molecules,into suitable cells. The term refers to both stable and transient uptakeof the genetic material.

The term “transduction” denotes the delivery of a DNA molecule to arecipient cell either in vivo or in vitro, via a replication-defectiveviral vector, such as via a recombinant AAV virion.

By “muscle cell” or “tissue” is meant a cell or group of cells derivedfrom muscle, including but not limited to cells and tissue derived fromskeletal muscle; smooth muscle, e.g., from the digestive tract, urinarybladder and blood vessels; and cardiac muscle. The term captures musclecells both in vitro and in vivo. Thus, for example, an isolatedcardiomyocyte would constitute a “muscle cell” for purposes of thepresent invention, as would a muscle cell as it exists in muscle tissuepresent in a subject in vivo. The term also encompasses bothdifferentiated and nondifferentiated muscle cells, such as myocytes,myotubes, myoblasts, cardiomyocytes and cardiomyoblasts.

The term “heterologous” as it relates to nucleic acid sequences such asgene sequences and control sequences, denotes sequences that are notnormally joined together, and/or are not normally associated with aparticular cell. Thus, a “heterologous” region of a nucleic acidconstruct or a vector is a segment of nucleic acid within or attached toanother nucleic acid molecule that is not found in association with theother molecule in nature. For example, a heterologous region of anucleic acid construct could include a coding sequence flanked bysequences not found in association with the coding sequence in nature.Another example of a heterologous coding sequence is a construct inwhich the coding sequence itself is not found in nature (e.g., syntheticsequences having codons different from the native gene). Similarly, acell transformed with a construct which is not normally present in thecell would be considered heterologous for purposes of this invention.Allelic variation or naturally occurring mutational events do not giverise to heterologous DNA, as used herein.

By “DNA” is meant a polymeric form of deoxyribonucleotides (adenine,guanine, thymine, or cytosine) in double-stranded or single-strandedform, either relaxed and supercoiled. This term refers only to theprimary and secondary structure of the molecule, and does not limit itto any particular tertiary forms. Thus, this term includes single- anddouble-stranded DNA found, inter alia, in linear DNA molecules (e.g.,restriction fragments), viruses, plasmids, and chromosomes. Indiscussing the structure of particular DNA molecules, sequences may bedescribed herein according to the normal convention of giving only thesequence in the 5′ to 3′ direction along the nontranscribed strand ofDNA (i.e., the strand having the sequence homologous to the mRNA). Theterm captures molecules that include the four bases adenine, guanine,thymine, or cytosine, as well as molecules that include base analogueswhich are known in the art.

A “gene” or “coding sequence” or a sequence which “encodes” a particularprotein, is a nucleic acid molecule which is transcribed (in the case ofDNA) and translated (in the case of mRNA) into a polypeptide in vitro orin vivo when placed under the control of appropriate regulatorysequences. The boundaries of the gene are determined by a start codon atthe 5′ (amino) terminus and a translation stop codon at the 3′ (carboxy)terminus. A gene can include, but is not limited to, cDNA fromprokaryotic or eukaryotic mRNA, genomic DNA sequences from prokaryoticor eukaryotic DNA, and even synthetic DNA sequences. A transcriptiontermination sequence will usually be located 3′ to the gene sequence.

The term “control elements” refers collectively to promoter regions,polyadenylation signals, transcription termination sequences, upstreamregulatory domains, origins of replication, internal ribosome entrysites (“IRES”), enhancers, and the like, which collectively provide forthe replication, transcription and translation of a coding sequence in arecipient cell. Not all of these control elements need always be presentso long as the selected coding sequence is capable of being replicated,transcribed and translated in an appropriate host cell.

The term “promoter region” is used herein in its ordinary sense to referto a nucleotide region comprising a DNA regulatory sequence, wherein theregulatory sequence is derived from a gene which is capable of bindingRNA polymerase and initiating transcription of a downstream(3′-direction) coding sequence.

“Operably linked” refers to an arrangement of elements wherein thecomponents so described are configured so as to perform their usualfunction. Thus, control elements operably linked to a coding sequenceare capable of effecting the expression of the coding sequence. Thecontrol elements need not be contiguous with the coding sequence, solong as they function to direct the expression thereof. Thus, forexample, intervening untranslated yet transcribed sequences can bepresent between a promoter sequence and the coding sequence and thepromoter sequence can still be considered “operably linked” to thecoding sequence.

For the purpose of describing the relative position of nucleotidesequences in a particular nucleic acid molecule throughout the instantapplication, such as when a particular nucleotide sequence is describedas being situated “upstream,” “downstream,” “3′,” or “5′” relative toanother sequence, it is to be understood that it is the position of thesequences in the “sense” or “coding” strand of a DNA molecule that isbeing referred to as is conventional in the art.

“Homology” refers to the percent of identity between two polynucleotideor two polypeptide moieties. The correspondence between the sequencefrom one moiety to another can be determined by techniques known in theart. For example, homology can be determined by a direct comparison ofthe sequence information between two polypeptide molecules by aligningthe sequence information and using readily available computer programs.Alternatively, homology can be determined by hybridization ofpolynucleotides under conditions which form stable duplexes betweenhomologous regions, followed by digestion with single-stranded-specificnuclease(s), and size determination of the digested fragments. Two DNA,or two polypeptide sequences are “substantially homologous” to eachother when at least about 80%, preferably at least about 90%, and mostpreferably at least about 95% of the nucleotides or amino acids matchover a defined length of the molecules, as determined using the methodsabove.

By “mammalian subject” is meant any member of the class Mammaliaincluding, without limitation, humans and nonhuman primates such aschimpanzees and other apes and monkey species; farm animals such ascattle, sheep, pigs, goats and horses; domestic mammals such as dogs andcats; laboratory animals including rodents such as mice, rats and guineapigs, and the like. The term does not denote a particular age or sex.Thus, adult and newborn subjects, as well as fetuses, whether male orfemale, are intended to be covered.

B. General Methods

The present invention provides for the successful transfer of a selectedgene to a muscle cell using recombinant AAV virions. The method allowsfor the direct, in vivo injection of recombinant AAV virions into muscletissue, e.g., by intramuscular injection, as well as for the in vitrotransduction of muscle cells which can subsequently be introduced into asubject for treatment. The invention also provides for secretion of theproduced protein in vivo, from transduced muscle cells, such thatsystemic delivery can be achieved.

Muscle provides a desirable target for gene therapy since muscle cellsare readily accessible and nondividing. However, the present inventionalso finds use with nondifferentiated muscle cells, such as myoblasts,which can be transduced in vitro, and subsequently introduced into asubject.

Since muscle has ready access to the circulatory system, a proteinproduced and secreted by muscle cells and tissue in vivo will enter thebloodstream for systemic delivery. Furthermore, since sustained,therapeutic levels of protein secretion from muscle is achieved in vivousing the present invention, repeated parenteral delivery is avoided orreduced in frequency such that therapy can be accomplished using onlyone or few injections. Thus, the present invention provides significantadvantages over prior gene delivery methods.

The recombinant AAV virions of the present invention, including the DNAof interest, can be produced using standard methodology, known to thoseof skill in the art. The methods generally involve the steps of (1)introducing an AAV expression vector into a producer cell.; (2)introducing an AAV helper construct into the producer cell, where thehelper construct includes AAV coding regions capable of being expressedin the producer cell to complement AAV helper functions missing from theAAV vector; (3) introducing one or more helper viruses and/or accessoryfunction vectors into the producer cell, wherein the helper virus and/oraccessory function vectors provide accessory functions capable ofsupporting efficient recombinant AAV (“rAAV”) virion production in thecell; and (4) culturing the producer cell to produce rAAV virions. TheAAV expression vector, AAV helper construct and the helper virus oraccessory function vector(s) can be introduced into the producer cell,either simultaneously or serially, using standard transfectiontechniques.

1. AAV Expression Vectors

AAV expression vectors are constructed using known techniques to atleast provide as operatively linked components in the direction oftranscription, control elements including a transcriptional initiationregion, the DNA of interest and a transcriptional termination region.The control elements are selected to be functional in a mammalian musclecell. The resulting construct which contains the operatively linkedcomponents is bounded (5′ and 3′) with functional AAV ITR sequences.

The nucleotide sequences of AAV ITR regions are known. See, e.g., Kotin,R. M. (1994) Human Gene Therapy 5:793-801; Berns, K. I. “Parvoviridaeand their Replication” in Fundamental Virology, 2nd Edition, (B. N.Fields and D. M. Knipe, eds.) for the AAV-2 sequence. AAV ITRs used inthe vectors of the invention need not have a wild-type nucleotidesequence, and may be altered, e.g., by the insertion, deletion orsubstitution of nucleotides. Additionally, AAV ITRs may be derived fromany of several AAV serotypes, including without limitation, AAV-1,AAV-2, AAV-3, AAV-4, AAV-5, AAVX7, etc. Furthermore, 5′ and 3′ ITRswhich flank a selected nucleotide sequence in an AAV expression vectorneed not necessarily be identical or derived from the same AAV serotypeor isolate, so long as they function as intended, i.e., to allow forpackaging of virions.

Suitable DNA molecules for use in AAV vectors will be less than about 5kilobases (kb) in size and will include, for example, a gene thatencodes a protein that is defective or missing from a recipient subjector a gene that encodes a protein having a desired biological ortherapeutic effect (e.g., an antibacterial, antiviral or antitumorfunction).

Suitable DNA molecules include, but are not limited to, those encodingfor proteins used for the treatment of endocrine, metabolic,hematologic, cardiovascular, neurologic, musculoskeletal, urologic,pulmonary and immune disorders, including such disorders as inflammatorydiseases, autoimmune, chronic and infectious diseases, such as AIDS,cancer, hypercholestemia, insulin disorders such as diabetes, growthdisorders, various blood disorders including various anemias,thalassemias and hemophilia; genetic defects such as cystic fibrosis,Gaucher's Disease, Hurler's Disease, adenosine deaminase (ADA)deficiency, emphysema, or the like.

To exemplify the invention, the gene encoding erythropoietin (EPO) canbe used. EPO is a glycoprotein hormone produced in fetal liver and adultkidney which acts on progenitor cells in the bone marrow and otherhematopoietic tissue to stimulate the formation of red blood cells.Genes encoding human and other mammalian EPO have been cloned, sequencedand expressed, and show a high degree of sequence homology in the codingregion across species. Wen et al. (1993) Blood 82:1507-1516. Thesequence of the gene encoding native human EPO, as well as methods ofobtaining the same, are described in, e.g., U.S. Pat. Nos. 4,954,437 and4,703,008, incorporated herein by reference in their entirety, as wellas in Jacobs et al. (1985) Nature 313:806-810; Lin et al. (1985) Proc.Natl. Acad. Sci. USA 82:7580; International Publication Number WO85/02610; and European Patent Publication Number 232,034 B1. Inaddition, the sequences of the genes encoding native feline, canine andporcine EPO are known and readily available (GenBank Accession Nos.:L10606; L13027; and L10607, respectively), and the sequence of the geneencoding monkey (Macaca mulatta) is also known and available (GenBankAccession No.: L10609). The term “EPO” as used herein refers to thenative, full-length secreted form of EPO, as well as to analogs orderivatives thereof comprising single or multiple amino acidsubstitutions, deletions or additions which retain EPO function oractivity. In this regard, a number of small peptides have beenidentified which bind to and activate the receptor for EPO. Wrighton etal. (1996) Science 273:458-463; Livnah et al. (1996) Science273:464-471. The recombinant AAV virions described herein which includea gene encoding EPO, or encoding an analog or derivative thereof havingthe same function, are particularly useful in the treatment of blooddisorders characterized by defective red blood cell formation, such asin the treatment of anemia. Increased red blood cell production due tothe production of EPO can be readily determined by an appropriateindicator, such as by comparing hematocrit measurements pre- andpost-treatment, measuring increases in red blood cell count, hemoglobinconcentration, or in reticulocyte counts. As described above, the EPOgene is flanked by AAV ITRs.

Alternatively, a nucleotide sequence encoding the lysosomal enzyme acidα-glucosidase (GAA) can be used. GAA functions to cleave α-1,4 and α-1,6linkages of lysosomal glycogen to release monosaccharides. The sequenceof the gene encoding human GAA, as well as methods of obtaining thesame, have been previously described (GenBank Accession Numbers: M34424and Y00839; Martiniuk et al. (1990) DNA Cell Biol. 9:85-94; Martiniuk etal. (1986) Proc. Natl. Acad. Sci. USA 83:9641-9644; Hoefsloot et al.(1988) Eur. Mol. Biol. Organ. 7:1697-1704). Thus, the recombinant AAVvirions described herein can include a nucleotide sequence encoding GAA,or encoding an analog or derivative thereof having GAA activity.

The selected nucleotide sequence, such as EPO or another gene ofinterest, is operably linked to control elements that direct thetranscription or expression thereof in the subject in vivo. Such controlelements can comprise control sequences normally associated with theselected gene. Alternatively, heterologous control sequences can beemployed. Useful heterologous control sequences generally include thosederived from sequences encoding mammalian or viral genes. Examplesinclude, but are not limited to, the SV40 early promoter; mouse mammarytumor virus LTR promoter; adenovirus major late promoter (Ad MLP);herpes simplex virus (HSV) promoters; a cytomegalovirus (CMV) promotersuch as the CMV immediate early promoter region (CMVIE); a rous sarcomavirus (RSV) promoter; synthetic promoters; hybrid promoters; and thelike. In addition, sequences derived from nonviral genes, such as themurine metallothionein gene, will also find use herein. Such promotersequences are commercially available from, e.g., Stratagene (San Diego,Calif.).

For purposes of the present invention, control elements, such asmuscle-specific and inducible promoters, enhancers and the like, will beof particular use. Such control elements include, but are not limitedto, those derived from the actin and myosin gene families, such as fromthe myoD gene family (Weintraub et al. (1991) Science 251:761-766); themyocyte-specific enhancer binding factor MEF72 (Cserjesi and Olson(1991) Mol. Cell Biol. 11:4854-4862); control elements derived from thehuman skeletal actin gene (Muscat et al. (1987) Mol. Cell Biol.7:4089-4099) and the cardiac actin gene; muscle creatine kinase sequenceelements (Johnson et al. (1989) Mol. Cell Biol. 9:3393-3399) and themurine creatine kinase enhancer (mCK) element; control elements derivedfrom the skeletal fast-twitch troponin C gene, the slow-twitch cardiactroponin C gene and the slow-twitch troponin I gene; hypoxia-induciblenuclear factors (Semenza et al. (1991) Proc. Natl. Acad. Sci. USA88:5680-5684; Semenza et al. J. Biol. Chem. 269:23757-23763);steroid-inducible elements and promoters, such as the glucocorticoidresponse element (GRE) (Mader and White (1993) Proc. Natl. Acad. Sci.USA 90:5603-5607); the fusion consensus element for RU486 induction;elements that provide for tetracycline regulated gene expression (Dhawanet al. (1995) Somat. Cell. Mol. Genet. 21:233-240; Shockett et al.(1995) Proc. Natl. Acad. Sci. USA 92:6522-6526; and inducible, synthetichumanized promoters (Rivera et al. (1996) Nature Med. 2:1028-1032).

These and other regulatory elements can be tested for potential in vivoefficacy using the in vitro myoblast model, which mimics quiescent invivo muscle physiology, described in the examples below.

The AAV expression vector which harbors the DNA molecule of interestbounded by AAV ITRs, can be constructed by directly inserting theselected sequence(s) into an AAV genome which has had the major AAV openreading frames (“ORFs”) excised therefrom. Other portions of the AAVgenome can also be deleted, so long as a sufficient portion of the ITRsremain to allow for replication and packaging functions. Such constructscan be designed using techniques well known in the art. See, e.g., U.S.Pat. Nos. 5,173,414 and 5,139,941; International Publication Nos. WO92/01070 (published Jan. 23, 1992) and WO 93/03769 (published Mar. 4,1993); Lebkowski et al. (1988) Molec. Cell. Biol. 8:3988-3996; Vincentet al. (1990) Vaccines 90 (Cold Spring Harbor Laboratory Press); Carter,B. J. (1992) Current Opinion in Biotechnology 3:533-539; Muzyczka, N.(1992) Current Topics in Microbiol. and Immunol. 158:97-129; Kotin, R.M. (1994) Human Gene Therapy 5:793-801; Shelling and Smith (1994) GeneTherapy 1:165-169; and Zhou et al. (1994) J. Exp. Med. 179:1867-1875.

Alternatively, AAV ITRs can be excised from the viral genome or from anAAV vector containing the same and fused 5′ and 3′ of a selected nucleicacid construct that is present in another vector using standard ligationtechniques, such as those described in Sambrook et al., supra. Forexample, ligations can be accomplished in 20 mM Tris-Cl pH 7.5, 10 mMMgCl₂, 10 mM DTT, 33 μg/ml BSA, 10 mM-50 mM NaCl, and either 40 μM ATP,0.01-0.02 (Weiss) units T4 DNA ligase at 0° C. (for “sticky end”ligation) or 1 mM ATP, 0.3-0.6 (Weiss) units T4 DNA ligase at 14° C.(for “blunt end” ligation). Intermolecular “sticky end” ligations areusually performed at 30-100 μg/ml total DNA concentrations (5-100 nMtotal end concentration). AAV vectors which contain ITRs have beendescribed in, e.g., U.S. Pat. No. 5,139,941. In particular, several AAVvectors are described therein which are available from the American TypeCulture Collection (“ATCC”) under Accession Numbers 53222, 53223, 53224,53225 and 53226.

Additionally, chimeric genes can be produced synthetically to includeAAV ITR sequences arranged 5′ and 3′ of one or more selected nucleicacid sequences. Preferred codons for expression of the chimeric genesequence in mammalian muscle cells can be used. The complete chimericsequence is assembled from overlapping oligonucleotides prepared bystandard methods. See, e.g., Edge, Nature (1981) 292:756; Nambair et al.Science (1984) 223:1299; Jay et al. J. Biol. Chem. (1984) 259:6311.

In order to produce rAAV virions, an AAV expression vector is introducedinto a suitable producer cell using known techniques, such as bytransfection. A number of transfection techniques are generally known inthe art. See, e.g., Graham et al. (1973) Virology, 52:456, Sambrook etal. (1989) Molecular Cloning, a laboratory manual, Cold Spring HarborLaboratories, New York, Davis et al. (1986) Basic Methods in MolecularBiology, Elsevier, and Chu et al. (1981) Gene 13:197. Particularlysuitable transfection methods include calcium phosphate co-precipitation(Graham et al. (1973) Virol. 52:456-467), direct micro-injection intocultured cells (Capecchi, M. R. (1980) Cell 22:479-488), electroporation(Shigekawa et al. (1988) BioTechniques 6:742-751), liposome mediatedgene transfer (Mannino et al. (1988) BioTechniques 6:682-690),lipid-mediated transduction (Felgner et al. (1987) Proc. Natl. Acad.Sci. USA 84:7413-7417), and nucleic acid delivery using high-velocitymicroprojectiles (Klein et al. (1987) Nature 327:70-73).

For the purposes of the invention, suitable producer cells for producingrAAV virions include microorganisms, yeast cells, insect cells, andmammalian cells, that can be, or have been, used as recipients of aheterologous DNA molecule. The term includes the progeny of the originalcell which has been transfected. Thus, a “producer cell” as used hereingenerally refers to a cell which has been transfected with an exogenousDNA sequence. Cells from the stable human cell line, 293 (readilyavailable through, e.g., the American Type Culture Collection underAccession Number ATCC CRL1573) are preferred in the practice of thepresent invention. Particularly, the human cell line 293 is a humanembryonic kidney cell line that has been transformed with adenovirustype-5 DNA fragments (Graham et al. (1977) J. Gen. Virol. 36:59), andexpresses the adenoviral E1a and E1b genes (Aiello et al. (1979)Virology 94:460). The 293 cell line is readily transfected, and providesa particularly convenient platform in which to produce rAAV virions.

2. AAV Helper Functions

Producer cells containing the above-described AAV expression vectorsmust be rendered capable of providing AAV helper functions in order toreplicate and encapsidate the nucleotide sequences flanked by the AAVITRs to produce rAAV virions. AAV helper functions are generallyAAV-derived coding sequences which can be expressed to provide AAV geneproducts that, in turn, function in trans for productive AAVreplication. AAV helper functions are used herein to complementnecessary AAV functions that are missing from the AAV expressionvectors. Thus, AAV helper functions include one, or both of the majorAAV ORFs, namely the rep and cap coding regions, or functionalhomologues thereof.

By “AAV rep coding region” is meant the art-recognized region of the AAVgenome which encodes the replication proteins Rep 78, Rep 68, Rep 52 andRep 40. These Rep expression products have been shown to possess manyfunctions, including recognition, binding and nicking of the AAV originof DNA replication, DNA helicase activity and modulation oftranscription from AAV (or other heterologous) promoters. The Repexpression products are collectively required for replicating the AAVgenome. For a description of the AAV rep coding region, see, e.g.,Muzyczka, N. (1992) Current Topics in Microbiol. and Immunol.158:97-129; and Kotin, R. M. (1994) Human Gene Therapy 5:793-801.Suitable homologues of the AAV rep coding region include the humanherpesvirus 6 (HHv-6) rep gene which is also known to mediate AAV-2 DNAreplication (Thomson et al. (1994) Virology 204:304-311).

By “AAV cap coding region” is meant the art-recognized region of the AAVgenome which encodes the capsid proteins VP1, VP2, and VP3, orfunctional homologues thereof. These cap expression products are thecapsid proteins which are collectively required for packaging the viralgenome. For a description of the AAV cap coding region, see, e.g.,Muzyczka, N. and Kotin, R. M. (supra).

AAV helper functions are introduced into the producer cell bytransfecting the cell with an AAV helper construct either prior to, orconcurrently with, the transfection of the AAV expression vector. AAVhelper constructs are thus used to provide at least transient expressionof AAV rep and/or cap genes to complement missing AAV functions that arenecessary for productive AAV infection. AAV helper constructs lack AAVITRs and can neither replicate nor package themselves. These constructscan be in the form of a plasmid, phage, transposon, cosmid, virus, orvirion. A number of AAV helper constructs have been described, such asthe commonly used plasmids pAAV/Ad and pIM29+45 which encode both Repand Cap expression products. See, e.g., Samulski et al. (1989) J. Virol.63:3822-3828; and McCarty et al. (1991) J. Virol. 65:2936-2945. A numberof other vectors have been described which encode Rep and/or Capexpression products. See, e.g., U.S. Pat. No. 5,139,941.

Both AAV expression vectors and AAV helper constructs can be constructedto contain one or more optional selectable markers. Suitable markersinclude genes which confer antibiotic resistance or sensitivity to,impart color to, or change the antigenic characteristics of those cellswhich have been transfected with a nucleic acid construct containing theselectable marker when the cells are grown in an appropriate selectivemedium. Several selectable marker genes that are useful in the practiceof the invention include the hygromycin B resistance gene (encodingAminoglycoside phosphotranferase (APH)) that allows selection inmammalian cells by conferring resistance to G418 (available from Sigma,St. Louis, Mo.). Other suitable markers are known to those of skill inthe art.

3. Accessory Functions

The producer cell must also be rendered capable of providing non AAVderived functions, or “accessory functions,” in order to produce rAAVvirions. Accessory functions are non AAV derived viral and/or cellularfunctions upon which AAV is dependent for its replication. Thus,accessory functions include at least those non AAV proteins and RNAsthat are required in AAV replication, including those involved inactivation of AAV gene transcription, stage specific AAV mRNA splicing,AAV DNA replication, synthesis of Cap expression products and AAV capsidassembly. Viral-based accessory functions can be derived from any of theknown helper viruses.

Particularly, accessory functions can be introduced into and thenexpressed in producer cells using methods known to those of skill in theart. Commonly, accessory functions are provided by infection of theproducer cells with an unrelated helper virus. A number of suitablehelper viruses are known, including adenoviruses; herpesviruses such asherpes simplex virus types 1 and 2; and vaccinia viruses. Nonviralaccessory functions will also find use herein, such as those provided bycell synchronization using any of various known agents. See, e.g.,Buller et al. (1981) J. Virol. 40:241-247; McPherson et al. (1985)Virology 147:217-222; Schlehofer et al. (1986) Virology 152:110-117.

Alternatively, accessory functions can be provided using an accessoryfunction vector. Accessory function vectors include nucleotide sequencesthat provide one or more accessory functions. An accessory functionvector is capable of being introduced into a suitable producer cell inorder to support efficient AAV virion production in the cell. Accessoryfunction vectors can be in the form of a plasmid, phage, transposon orcosmid. Accessory vectors can also be in the form of one or morelinearized DNA or RNA fragments which, when associated with theappropriate control elements and enzymes, can be transcribed orexpressed in a producer cell to provide accessory functions.

Nucleic acid sequences providing the accessory functions can be obtainedfrom natural sources, such as from the genome of an adenovirus particle,or constructed using recombinant or synthetic methods known in the art.In this regard, adenovirus-derived accessory functions have been widelystudied, and a number of adenovirus genes involved in accessoryfunctions have been identified and partially characterized. See, e.g.,Carter, B. J. (1990) “Adeno-Associated Virus Helper Functions,” in CRCHandbook of Parvoviruses, vol. I (P. Tijssen, ed.), and Muzyczka, N.(1992) Curr. Topics. Microbiol. and Immun. 158:97-129. Specifically,early adenoviral gene regions E1a, E2a, E4, VAI RNA and, possibly, E1bare thought to participate in the accessory process. Janik et al. (1981)Proc. Natl. Acad. Sci. USA 78:1925-1929. Herpesvirus-derived accessoryfunctions have been described. See, e.g., Young et al. (1979) Prog. Med.Virol. 25:113. Vaccinia virus-derived accessory functions have also beendescribed. See, e.g., Carter, B. J. (1990), supra., Schlehofer et al.(1986) Virology 152:110-117.

As a consequence of the infection of the producer cell with a helpervirus, or transfection of the producer cell with an accessory functionvector, accessory functions are expressed which transactivate the AAVhelper construct to produce AAV Rep and/or Cap proteins. The Repexpression products excise the recombinant DNA (including the DNA ofinterest) from the AAV expression vector. The Rep proteins also serve toduplicate the AAV genome. The expressed Cap proteins assemble intocapsids, and the recombinant AAV genome is packaged into the capsids.Thus, productive AAV replication ensues, and the DNA is packaged intorAAV virions.

Following recombinant AAV replication, rAAV virions can be purified fromthe producer cell using a variety of conventional purification methods,such as CsCl gradients. Further, if infection is employed to express theaccessory functions, residual helper virus can be inactivated, usingknown methods. For example, adenovirus can be inactivated by heating totemperatures of approximately 60° C. for, e.g., 20 minutes or more. Thistreatment effectively inactivates only the helper virus since AAV isextremely heat stable while the helper adenovirus is heat labile.

The resulting rAAV virions are then ready for use for DNA delivery, suchas in gene therapy applications, for the production of transgenicanimals, in vaccination, and particularly for the delivery of genes to avariety of muscle cell types.

4. In vitro and In vivo Delivery of rAAV Virions

Generally, rAAV virions are introduced into a muscle cell using eitherin vivo or in vitro transduction techniques. If transduced in vitro, thedesired recipient muscle cell will be removed from the subject,transduced with rAAV virions and reintroduced into the subject.Alternatively, syngeneic or xenogeneic muscle cells can be used wherethose cells will not generate an inappropriate immune response in thesubject.

Suitable methods for the delivery and introduction of transduced cellsinto a subject have been described. For example, cells can be transducedin vitro by combining recombinant AAV virions with muscle cells e.g., inappropriate media, and screening for those cells harboring the DNA ofinterest using conventional techniques such as Southern blots and/orPCR, or by using selectable markers. Transduced cells can then beformulated into pharmaceutical compositions, described more fully below,and the composition introduced into the subject by various techniques,such as by intramuscular, intravenous, subcutaneous and intraperitonealinjection, or by injection into smooth and cardiac muscle, using e.g., acatheter.

For in vivo delivery, the rAAV virions will be formulated intopharmaceutical compositions and will generally be administeredparenterally, e.g., by intramuscular injection directly into skeletal orcardiac muscle.

Pharmaceutical compositions will comprise sufficient genetic material toproduce a therapeutically effective amount of the protein of interest,i.e., an amount sufficient to reduce or ameliorate symptoms of thedisease state in question or an amount sufficient to confer the desiredbenefit. The pharmaceutical compositions will also contain apharmaceutically acceptable excipient. Such-excipients include anypharmaceutical agent that does not itself induce the production ofantibodies harmful to the individual receiving the composition, andwhich may be administered without undue toxicity. Pharmaceuticallyacceptable excipients include, but are not limited to, liquids such aswater, saline, glycerol and ethanol. Pharmaceutically acceptable saltscan be included therein, for example, mineral acid salts such ashydrochlorides, hydrobromides, phosphates, sulfates, and the like; andthe salts of organic acids such as acetates, propionates, malonates,benzoates, and the like. Additionally, auxiliary substances, such aswetting or emulsifying agents, pH buffering substances, and the like,may be present in such vehicles. A thorough discussion ofpharmaceutically acceptable excipients is available in REMINGTON'SPHARMACEUTICAL SCIENCES (Mack Pub. Co., N.J. 1991).

Appropriate doses will depend on the mammal being treated (e.g., humanor nonhuman primate or other mammal), age and general condition of thesubject to be treated, the severity of the condition being treated, theparticular therapeutic protein in question, its mode of administration,among other factors. An appropriate effective amount can be readilydetermined by one of skill in the art.

Thus, a “therapeutically effective amount” will fall in a relativelybroad range that can be determined through clinical trials. For example,for in vivo injection, i.e., injection directly to skeletal or cardiacmuscle, a therapeutically effective dose will be on the order of fromabout 10⁶ to 10¹⁵ of the rAAV virions, more preferably 10⁸ to 10¹⁴ rAAVvirions. For in vitro transduction, an effective amount of rAAV virionsto be delivered to muscle cells will be on the order of 10⁸ to 10¹³ ofthe rAAV virions. The amount of transduced cells in the pharmaceuticalcompositions will be from about 10⁴ to 10¹⁰ muscle cells, morepreferably 10⁵ to 10⁸ muscle cells. When the transduced cells areintroduced to vascular smooth muscle, a lower dose may be appropriate.Other effective dosages can be readily established by one of ordinaryskill in the art through routine trials establishing dose responsecurves.

Dosage treatment may be a single dose schedule or a multiple doseschedule. Moreover, the subject may be administered as many doses asappropriate. One of skill in the art can readily determine anappropriate number of doses.

C. Experimental

Below are examples of specific embodiments for carrying out the presentinvention. The examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.

Efforts have been made to ensure accuracy with respect to numbers used(e.g., amounts, temperatures, etc.), but some experimental error anddeviation should, of course, be allowed for.

Materials and Methods

Vector constructs

A. Construction of p1909adhlacZ.

Plasmid p1909adhlacZ was used as the helper construct in the followingexamples and was constructed from plasmid pWadhlacZ. Plasmid pWadhlacZwas constructed by partially digesting plasmid pUC119 (GeneBankReference Name: U07649, GeneBank Accession Number: U07649) with AflIIIand BspHI, blunt-end modifying with the klenow enzyme, and then ligatingto form a circular 1732 bp plasmid containing the bacterial origin andthe amp gene only (the polylinker and F1 origin was removed). Theblunted and ligated AflIII and BspHI junction forms a unique NspI site.The 1732 bp plasmid was cut with NspI, blunt-end modified with T4polymerase, and a 20 bp HinDIII-HinCII fragment (blunt-end modified withthe klenow enzyme) obtained from the pUC119 polylinker was ligated intothe blunted NspI site of the plasmid. The HinDIII site from the bluntedpolylinker was regenerated, and then positioned adjacent to thebacterial origin of replication. The resulting plasmid was then cut atthe unique PstI/Sse8387I site, and an Sse8387I-PvuII-Sse8387Ioligonucleotide, having the sequence: 5′-GGCAGCTGCCTGCA-3′ (SEQ IDNO.:1), was ligated therein. The remaining unique BapHI site was cut,blunt-end modified with klenow enzyme, and an AscI linkeroligonucleotide, having the sequence: 5′-GAAGGCGCGCCTTC-3′ (SEQ IDNO.:2) was ligated therein, eliminating the BspHI site. The resultingplasmid was called pWee.

In order to create the pWadhlacZ construct, a CMVlacZ expressioncassette (comprising a nucleotide sequence flanked 5′ and 3′ by AAVITRS, containing the following elements: a CMV promoter, the hGH 1stintron, an adhlacz fragment and an SV40 early polyadenylation site) wasinserted into the unique PvuII site of pWee using multiple steps suchthat the CMV promoter was arranged proximal to the bacterial amp gene ofpWee.

More particularly, a CMVlacZ expression cassette was derived from theplasmid psub201CMV, which was constructed as follows. An oligonucleotideencoding the restriction enzyme sites:NotI-MluI-SnaBI-AgeI-BstBI-BssHII-NcoI-HpaI-BspEI-PmlI-RsrII-NotI andhaving the following nucleotide sequence:

5′GCGGCCGCACGCGTACGTACCGGTTCGAAGCGCGCACGGCCGACCATGGTTAAC (SEQ ID No: 3)TCCGGACACGTGCGGACCGCGGCCGC-3′

was synthesized and cloned into the blunt-end modified KasI-EarI site(partial) of pUC119 to provide a 2757 bp vector fragment. A 653 bpSpeI-SacII fragment containing a nucleotide sequence encoding a CMVimmediate early promoter was cloned into the SnaBI site of the 2757 bpvector fragment. Further, a 269 bp PCR-produced BstBI-BstBI fragmentcontaining a nucleotide sequence encoding the hGH 1st intron which wasderived using the following primers:

5′-AAAATTCGAACCTGGGGAGAAACCAGAG-3′ (SEQ ID NO.: 4)3′-aaaattcgaacaggtaagcgcccctTTG-5′ (SEQ ID NO.: 5),

cloned into the BstBI site of the 2757 bp vector fragment, and a 135 bpHpaI-BamHI (blunt-end modified) fragment containing the SV40 earlypolyadenylation site from the pCMV-β plasmid (CLONETECH) was cloned intothe HpaI site of the subject vector fragment. The resulting constructwas then cut with NotI to provide a first CMV expression cassette.

Plasmid pW1909adhlacZ was constructed as follows. A 4723 bp SpeI-EcoRVfragment containing the AAV rep and cap encoding region was obtainedfrom the plasmid pGN1909 (ATCC Accession Number 69871). The pGN1909plasmid is a high efficiency AAV helper plasmid having AAV rep and capgenes with an AAV p5 promoter region that is arranged in the constructto be downstream from its normal position (in the wild type AAV genome)relative to the rep coding region. The 4723 bp fragment was blunt-endmodified, and AscI linkers were ligated to the blunted ends. Theresultant fragment was then ligated into the unique AscI site ofpWadhlacZ and oriented such that the AAV coding sequences were arrangedproximal to the bacterial origin of replication in the construct.

Plasmid pW1909adhlacZ includes the bacterial beta-galactosidase (β-gal)gene under the transcriptional control of the cytomegalovirus immediateearly promoter (CMVIE).

B. Construction of pW1909EPO.

Plasmid pW1909adhlacZ was modified to express human erythropoietin (EPO)by replacing the adhlacz gene with a 718 base pair PpuMI-NcoI fragmentof human EPO cDNA (Wen et al. (1993) Blood 5:1507-1516) and by cloning a2181 bp ClaI-EcoRI lacZ spacer fragment (noncoding) into the PmlI siteof the vector.

C. Construction of pAAV-GAA.

A plasmid containing the human lysosomal enzyme acid α-glucosidase (GAA)coding region was constructed as follows. A 3.2 kB cDNA clone containingthe coding sequence for human GAA beginning 207 bps downstream from theinitiation codon (GenBank Accession Numbers: M34424 and Y00839;Martiniuk et al. (1990) DNA Cell Biol. 9:85-94) was cloned into theEcoRI site of Bluescript KS (Stratagene). Additional 5′ sequence wasgenerated using polymerase chain reaction (PCR) with reverse-transcribedpoly-A mRNA (obtained from normal human fibroblasts) as the template.The 5′ primer was constructed with a KpnI restriction site and bps -3 to12 of the published sequence (Martiniuk et al. (1986) Proc. Natl. Acad.Sci. USA 83:9641-9644; Hoefsloot et al. (1988) Eur. Mol. Biol. Organ.7:1697-1704). The 3′ primer was synthesized from basepairs 1001 to 1018.Using KpnI and the unique internal StuI site at position 776, the PCRproduct was ligated to the partial cDNA to form the full-lengthGAA-encoding plasmid. The full length cDNA was truncated at a uniqueSphI restriction site, and cloned into the expression vector p1.1c toresult in the pAAV-GAA construct having the GAA coding region undertranscriptional control of the CMV-IE promoter.

The p1.1c expression vector was constructed as follows. pUC119 waspartially digested with KasI and EarI, and a 2713 bp vector fragmentcontaining the ampicillin resistance gene, the coli 1 origin ofreplication and the M13 origin of replication, was isolated, blunt endmodified, and ligated to a synthetic DNA polylinker encoding therestriction enzyme sitesNotI-MluI-SnaBI-AgeI-SfuI-BssHII-EagI-NCOI-PmeI-BspEI-PmlI-RsrII-NotI,and having the following nucleotide sequence:

5′-GCGGCCGCACGCGTTGTTAACAACCGGTTCGAAGCGCG (SEQ ID NO.: 6)CAGCGGCCGACCATGGGTTTAAACTCCGGAACCACGTGCGGACCGAGCGGCCGC-3′.

The ligation was conducted such that the MluI end of the polylinker wasligated to the KasI side of the plasmid. A 653 bp SpeI-SacII fragmentencoding the CMV immediate-early promoter, a 269 bp PCR-producedproduced SfuI-SfuI produced fragment encoding the hGH 1st intron(derived using the following primers:

5′-AAAATTCGAACAGGTAAGCGCCCCTTTG-3′ (SEQ ID NO.: 7) and (SEQ ID NO.: 8))3′-AAAATTCGAACCTGGGGAGAAACCAGAG-5′,

a 183 bp BssHII-BssHII polylinker fragment from pBluescript II SK-, anda 135 bp HpaI-BamHI (blunted) fragment containing the SV40 earlypolyadenylation site from pCMV-β (Stratagene), were cloned into theSnaBI, SfuI, BssHII, and PmeI sites, respectively, of the aforementionedplasmid. The orientation of the polylinker relative to the intron andpolyadenylation site was intron-polylinker (5′ SacI-3′KpnI)-polyadenylation site. The polylinker was further modified byremoving the 88 bp SacI-XhoI polylinker fragment and replacing it withthe following synthetic SacI to XhoI fragment encoding the restrictionenzyme sites SacI-ClaI-EcoRI-SmaI-BanrHI-XbaI-SalI-PstI-BstXI-EcoRV-BstXI-olmeganuclease-HinDIII-XhoI, having the followingnucleotide sequence:

5′-GAGCTCAATCGATTGAATTCCCCGGGGATCCTCTAGAGTCGACCTGCAGCCACT (SEQ ID NO.:9). GTGTTGGATATCCAACACACTGGTAGGGATAACAGGGTAATCTCGAG-3′

Viruses and Cell Lines

Adenovirus type 2 (Ad2), available from the American Type CultureCollection, ATCC, Catalogue Number VR846, was used as helper virus toencapsidate vectors.

The human 293 cell line (Graham et al. (1977) J. Gen. Virol. 36:59-72,available from the ATCC under Accession no. CRL1573), which hasadenovirus E1a and E1b genes stably integrated in its genome, wascultured in complete Dulbecco's modified Eagle's media (DMEM;Bio-Whitakker, Walkersville, Md.) containing 4.5 g/L glucose, 10%heat-inactivated fetal bovine serum (FBS; Hyclone, Logan, Utah), 2 mMglutamine, and 50 units/mL penicillin and 50 μg/mL streptomycin.

The C2C12 murine myoblast cell line, available from the ATCC, CatalogueNumber CRL1772, was cultured in DMEM with 20% fetal calf serum (FCS), 1%chick embryo extract and 5 μg/mL gentamicin.

Fetal human skeletal myoblasts (Clonetics) were cultured in Hams F-12human growth medium, containing 20% FCS and 5 μg/mL gentamicin.

The above cell lines were incubated at 37° C. in 5% CO₂, and wereroutinely tested and found free of mycoplasma contamination.

Production of Recombinant AAV Virions

Recombinant AAV virions were produced in human 293 cells as follows.Subconfluent 293 cells were cotransfected by standard calcium phosphateprecipitation (Wigler et al. (1979) Proc. Natl. Acad. Sci USA76:1373-1376) with one of the AAV vector/helper plasmid constructs,pW1909adhLacZ or pW1909EPO; or with pAAV-GAA and the pW1909 helperplasmid. After 6 hours, the transfected cells were infected with Ad2 infresh medium at a multiplicity of infection (MOI) of 2, and incubated at37° C. in 5% CO₂ for 70 hours prior to harvest. Pelleted cells werelysed in Tris buffer (10 mM Tris, 150 mM NaCl, pH 8.0) by three cyclesof freeze-thaw. The lysate was clarified of cell debris bycentrifugation at 12,000×g, and the crude-cell lysate was layered onto acesium chloride cushion for isopyknic gradient centrifugation.Recombinant AAV virions (rAAV-LacZ, rAAV-hEPO, or rAAV-hGAA virions)were extracted from the resulting gradient by isolating the fractionswith an average density of approximately 1.38 g/mL, resuspended in Hepesbuffered saline (HBS) containing 50 mM Hepes (pH 7.4) and 150 mM NaCl.The preparations were then heated at 56° C. for approximately 1 hour toinactivate Ad2.

Assay of rAAV by Dot-blot Hybridization

Recombinant AAV virions were DNase I digested, proteinase K treated,phenol-chloroform extracted, and DNA precipitated with sodiumacetate-glycogen (final concentrations=0.3 M sodium acetate and 160μg/mL, respectively). DNA samples were denatured (200 μL of 2× alkalinesolution (0.8 M NaOH, 20 mM EDTA) added to DNA sample) for 10 minutes,then added to appropriate wells in a dot-blot apparatus, and blottedonto wet Zeta Probe membrane (BioRad), by applying suction until wellswere empty. Then, 400 μL of 1× alkaline solution was added; after 5minutes, wells were emptied by suction. The membrane was rinsed in 2×SSC (Sambrook et al., supra) for 1 min, drained, air dried on filterpaper, then baked in vacuum at 80° C. for 30 min. The membrane was thenprehybridized for 30 min at 65° C. with 10 mL hybridization buffer (7%SDS, 0.25 M Sodium Phosphate, pH 7.2, 1 mM EDTA). Buffer was replacedwith 10 mL fresh solution, freshly boiled probe added, and hybridizedovernight at 65° C. The membrane was washed twice with 25 mL of wash-1buffer (5% SDS, 40 mM sodium phosphate, pH 7.2, 1 mM EDTA) for 20 min at65° C. and twice with wash-2 buffer (1% SDS, 40 mM sodium phosphate, pH7.2, 1 mM EDTA). The membrane was wrapped in plastic film, exposed toradiographic film, and appropriate dots excised from the membrane todetermine radioactivity by scintillation counting, and quantitated bycomparison with standards. Titers of rAAV virion were routinely in therange of approximately 10¹³ genomes/mL.

Assay for Contaminating Helper Adenovirus

Contaminating infectious adenovirus was assayed as follows. Samples fromthe purified rAAV virion stocks were added to 50% confluent 293 cells(cultured in 12 well dishes at 1×10⁵ cells/well), and the cultures werepassaged for 30 days (e.g., the cultures were split 1 to 5, every 3days) or until the culture exhibited 100% cytopathic effect (CPE) due toadenovirus infection. Cultures were examined daily for CPE, and the dayupon which each experimental culture showed 100% CPE was noted.Reference 293 cell cultures infected with a range of known amounts ofadenovirus type-2 (from 0 to 1×10⁷ plaque forming units (pfu)/culture)were also prepared and treated in the same manner. A standard curve wasthen prepared from the data obtained from the reference cultures, wherethe adenovirus pfu number was plotted against the day of 100% CPE. Thetiter of infectious adenovirus type-2 in each experimental culture wasthen readily obtained as determined from the standard curve. The limitof detection of the assay was 100 pfu/mL. The presence of wild-type AAVcontamination, analyzed by dot-blot hybridization, was approximately 7logs lower than recombinant virion concentration.

Differentiation of Myoblasts

C2C12 myoblasts were transduced either while actively dividing, or as adifferentiated cell culture. Differentiation was induced by placingsubconfluent myoblasts in murine differentiation medium (DMEM containing2% horse serum and standard concentrations of glutamine andpenicillin-streptomycin) for an interval of four days prior totransduction in order to induce myoblast fusion and formation ofdifferentiated myotubes.

Fetal human skeletal myoblasts were differentiated in humandifferentiation medium (DMEM containing 10% horse serum and 5 μg/mLgentamicin). Verification of differentiation was performed bymicroscopic analysis to determine the presence of multinucleatedmyotubes in culture.

EXAMPLE 1 Expression of rAAV-LacZ in Terminally Differentiated Adult RatCardiomyocytes

The ability of recombinant AAV virions to transduce terminallydifferentiated adult cardiomyocytes was established in vitro.Cardiomyocytes were harvested by coronary perfusion with collagenase ofadult rat hearts (Fischer 344, Harlan Sprague Dawley, Indianapolis,Ind.). Cardiomyocytes were grown on laminin-coated glass coverslips andexposed to rAAV-LacZ virions for 4 hours. After 72 hours, the cells werestained for β-galactosidase activity. AAV expression was detected byblue staining of the binucleated cells. These studies demonstrated theability of rAAV virions to transduce terminally differentiated cells.The transduction efficiency in vitro was 30% of adult cells at amultiplicity of infection (MOI) of 10⁴ genomes per cell.

EXAMPLE 2 Stability of rAAV-LacZ Expression In Vivo

Adult Fischer rats were used to analyze expression of transgenes invivo. Incremental doses of rAAV-LacZ virions were injected into the leftventricular apex of the heart using either a subxyphoid or lateralthoracotomy approach. More particularly, experimental animals wereanesthetized with Metofane followed by a subxyphoid incision to exposethe diaphragmatic surface of the heart. Apical cardiac injections wereperformed with a glass micropipette. Recombinant virion was diluted innormal saline and injected at a volume of 20-50 μL.

At varying times post-injection, hearts were harvested and examined forβ-galactosidase production and for the presence of infiltratingmononuclear cells. For 5-Bromo-4-chloro-3-indolyl β-D-galactosidehistochemical determination, frozen sections (6 μm) were fixed in 0.5%glutaraldehyde and stained for β-galactosidase activity as described(Sanes et al. (1986) “Use of Recombinant Retrovirus to StudyPost-Implantation Cell Lineage in Mouse Embryos,” EMBO J 5:3133-3142).Paraffin sections (5 μm) were stained with hematoxylin/eosin. Sectionswere examined for infiltrating mononuclear cells.

The above-described histochemical studies showed greater than 50%transduction of cardiomyocytes in the region of injection at each timepoint examined. Further, there was no inflammatory cell infiltrate notedduring the course of analysis. β-galactosidase staining was observed topersist in cardiac muscle for at least two months following genetransfer.

EXAMPLE 3 In vivo Transduction of Murine Skeletal Muscle using rAAV-LacZVirions

Recombinant AAV-LacZ virions were injected into muscle tissue of mice,and transduction assessed by β-gal activity. Particularly, in vivotransduction was performed by intramuscular (IM) injection ofrecombinant AAV virions into the skeletal muscle of healthy 6-8 week oldBalb/c mice (Jackson Laboratories, Bar Harbor, Me., SimonsenLaboratories, Gilroy, Calif., or Harlan Laboratories) under eitherMetofane (Pitman-Moore, Mundelein, Ill.) or ketamine-xylazineanesthesia. The mid-portion of each tibialis anterior muscle was exposedvia a 1 cm incision. Injections into the tibialis anterior were carriedout using a micro-capillary tube attached to a Hamilton syringe toadminister the following formulations at a depth of 2 mm:phosphate-buffered saline (PBS) alone (negative control); or PBScontaining either rAAV-LACZ virions or pW1909adhlacZ plasmids.

Tissue samples of tibialis anterior muscle, forelimb muscle, heart,brain and liver were obtained for analysis of β-galactosidaseexpression. One tibialis anterior muscle from each animal was processedfor cross-sectional β-galactosidase analysis, and total β-galactosidasewas determined from a crude homogenate of the other muscle using achemiluminescent assay.

For histochemical detection of β-galactosidase, muscle samples weresnap-frozen in dry ice-cooled isopentane, followed by serial transversesectioning (10 μm) and processing according to previously describedmethods (Sanes et al. (1986) EMBO J 5:3133-3142). The cross-sectionalarea of the tibialis anterior expressing β-galactosidase was determinedas follows: after counter-staining with nuclear fast red (Vector Labs),the X-gal stained tissue was digitally photographed and thecross-sectional area of stored images was determined using NIH Imagesoftware.

The GALACTO-LIGHT™ (Tropix, Bedford, Mass.) chemiluminescent reporterassay kit was used to detect total β-galactosidase activity in theentire tibialis anterior muscle. Standard curves were prepared fromknown amounts of purified β-galactosidase (Sigma, St. Louis, Mo.)resuspended in non-transduced muscle homogenate. β-galactosidaseactivity is expressed as either: nanograms of β-galactosidase,normalized for the entire muscle, minus background activity; or in termsof relative light units (RLU) as quantified by luminometer. Forelimbmuscle, cardiac muscle, brain tissue and liver samples were assayed inan identical fashion.

A. Time course of β-galactosidase expression.

A single intramuscular injection into the left and right tibialisanterior muscles (under direct vision) was used to deliver 8×10⁹rAAV-LacZ in a PBS vehicle. Four animals were injected with PBS alone.Animals were sacrificed at 2, 4, 8, 12, 24 and 32 weeks after injection,and the tibialis anterior muscle was excised and analyzed for thepresence of bacterial β-galactosidase (n=5 for each group) as describedabove.

Efficiency of the LacZ gene transfer was assessed by cross-sectionaltissue staining and chemiluminescent assay. As can be seen in FIG. 1 andin Table I below, gene expression persisted for at least 32 weeks. Inaddition, two weeks after injection of the recombinant virions, 18% ofthe muscle cross-sectional area expressed β-galactosidase, while at 32weeks, 24% of the muscle cross-sectional area expressed β-galactosidase.Negative control tibialis anterior muscle (obtained from the animalsinjected with PBS alone) showed no background staining. Muscleβ-galactosidase activity was also determined in the contralateralinjected muscle. This study also revealed persistent expression for atleast 32 weeks, in agreement with the cross-sectional fiber analysis(Table I). Further, the observed β-galactosidase was sustained withminimal inflammatory cell infiltrate. These data demonstrate thatrAAV-LacZ virion administration into muscle results in stable expressionof the transgene for at least 8 months.

Upon histological examination, positive staining filled the cytoplasm ofthe transduced myofibers and was observed through large contiguousportions of the muscle. Serial transverse sections revealed that bluestaining extended throughout the length of the muscle fiber.Diffraction-interference contrast microscopy revealed a cleardelineation between positively and negatively stained myofibers (FIG.2), suggesting that recombinant virion delivery was limited bystructural barriers such as epimyseal or perimyseal connective tissue.Homogenates prepared from brain, heart, liver, and forelimb muscledisplayed no β-galactosidase activity when compared with the backgroundactivity of the negative control animals.

TABLE I Time Course of β-galactosidase Expression Following SingleInjection with rAAV-LacZ. Percent cross- β-galactosidase sectional areaTime expression expressing β- (weeks) (ng/muscle) galactosidase (%)  21441 ± 458  18 ± 6   4 951 ± 176 20 ± 4   8 839 ± 436 24 ± 3  12 1878 ±521  29 ± 5  24 2579 ± 1165 22 ± 3  32 1242 ± 484  24 ± 3  The left andright tibialis anterior muscles were injected with 8 × 10⁹ rAAV-lacZ.One member of the pair of injected muscles was processed forβ-galactosidase expression (n = 5 ± SEM). The other muscle was processedfor histochemical detection of β-galactosidase and determination of thecross-sectional area of the tibialis anterior expressingβ-galactosidase. Mean cross-sectional areas ± SEM are shown.

B. Dose-response assay.

To determine the effective dose range for rAAV-LacZ in vivo, recombinantvirions were injected into the tibialis anterior muscle of 6-8 week oldBalb/c mice, and transduction assessed by β-galactosidase activity asmeasured by GLACTO-LIGHT™ relative light units (RLU). As can be seen inFIG. 3, at two weeks post-injection, the observed RLU ranged fromapproximately 0.2×10⁷ RLU/muscle (injected with 8×10⁹ rAAV-LacZ) toapproximately 1.1×10⁹ RLU/muscle (injected with 3.6×10¹¹ rAAV-LacZ).

The levels of expression of β-galactosidase measured in RLU correspondto the percentage of β-galactosidase -positive muscle fibers oncross-sectional analysis. For example, 0.2×10⁷ RLU corresponds toapproximately it β-galactosidase positive muscle fibers and 1.1×10⁹ RLUcorresponds to approximately 60% β-galactosidase positive muscle fibers.

C. Comparison of β-galactosidase expression efficiency.

A comparison of β-galactosidase expression efficiency obtained by invivo transduction of mice using either rAAV-LacZ virions, or plasmid DNAcontaining the same LacZ expression cassette (pW1909adhlacZ) was carriedout as follows. Either 8×10⁹ rAAV-LacZ, or 100 μg of pW1909adhlacZ, wasinjected into the tibialis anterior muscle of 6-8 week old Balb/c mice.Two weeks post-injection, β-galactosidase activity was assessed usingthe GALACTO-LIGHT™ chemiluminescent reporter assay kit, as describedabove. Administration of the recombinant virions resulted in 1441 ngβ-galactosidase/muscle (n=5), while administration of 100 μg of theplasmid DNA, a typical in vivo plasmid DNA dosage (Whalen et al. (1995)Hum. Gene Ther. 4:151-159), resulted in 12 ng β-galactosidase/muscle(n=4). This dosage of pW1909adhlacZ plasmid DNA is equivalent to2.2×10¹³ single stranded genomes, demonstrating that gene delivery bythe recombinant virions was substantially more efficient than deliveryof an equal molar quantity of vector DNA.

EXAMPLE 4 In vitro Transduction of Murine Myotubes and Myoblasts

In order to determine if differentiated cultured muscle cells areappropriate targets for recombinant AAV virion transduction, and toassess the ability of such cells to express a transduced gene, thefollowing study was carried out. Murine C2C12 cells were selected sincethese cells have been extensively studied as a model for mammalianmyogenesis (Blau et al. (1993) Trends Genet. 9:269-274), and can beinduced to differentiate by growth in reduced serum medium.

In the study, C2C12 myoblasts (dividing cells) were seeded in cellculture plates at a density of 2×10⁴ cells/cm², maintained in growthmedia (GM) until confluent, split, and then either cultured in GM orcultured for 5 days in murine DM. Differentiation was verified by themicroscopic presence of multinucleate myotubes, representing fusedmyoblasts (differentiated C2C12 cells).

The C2C12 myotubes and myoblasts were transduced in culture withpurified rAAV-hEPO virions at a MOI of 10⁵ in OptiMEM (Gibco BRL). Inthe myotube cultures, DM was added after virion adsorption. The culturemedia of the transduced cells was changed 24 hours prior to collectionof supernatants at 3, 8 and 14 days following transduction. Secretion ofhEPO was assessed by ELISA using the human erythropoietin Quantikine IVDkit (available from R and D Systems, Minneapolis, Minn.) according tomanufacturer's recommendations.

The results of the study show that hEPO is secreted from both thetransduced myotubes and myoblasts. The levels of hEPO secretionincreased in the myotubes over the first seven days post-transduction(FIG. 4). As can be seen by reference to FIG. 5, a dose-dependentincrease in the secretion of hEPO was also observed in the transducedC2C12 myotubes. Eight days post-transduction of the myotubes, hEPOlevels peaked at >3400 mU/mL. These data demonstrate that transductionwith rAAV-hEPO of both myotubes or myoblasts results in hEPO secretionby the transduced cells, and that in short-term myotube cultures, hEPOis synthesized and secreted in a dose-dependent manner.

EXAMPLE 5 In vitro Transduction of Human Myotubes Using rAAV-hEPOVirions

To determine if differentiated primary human muscle cells are able toexpress hEPO following transduction with rAAV-hEPO, the following studywas carried out. Primary fetal human skeletal myoblasts were seeded incell culture plates at a density of 2×10⁴ cells/cm², grown to confluencein appropriate growth media, and then cultured for 14 days in human DM.Differentiation was verified by microscopic examination formultinucleate cells. In vitro transduction was carried out by addingpurified rAAV-hEPO virions to the cultured myotubes in OptiMEM medium(Gibco BRL). DM was added to the cultures after virion adsorption.Control cultures were transduced with rAAV-LacZ.

Culture media was changed 24 hours prior to collection of supernatantsat day 3, 8 and 14 post transduction. Secreted EPO levels were assayedby ELISA as described above in Example 4.

As can be seen in FIG. 6, the transduced human myotubes secreted hEPOinto the culture in a dose-dependent manner. No detectable EPO activitywas measured in the control cultures. Secretion of EPO increased overthe 14-day interval post-transduction. These data demonstrate thatprimary human myotubes transduced by recombinant AAV virions are capableof expressing and secreting erythropoietin.

EXAMPLE 6 Systemic Delivery of Human Erythropoietin In vivo byIntramuscular Administration of rAAV-hEPO

Recombinant AAV virions encoding hEPO were administered to adult healthyBalb/c mice in vivo to determine if a systemic level of hEPO can beproduced, and a biological response obtained. At various time pointsafter administration, blood was obtained from the orbital venous plexusunder anesthesia. Serum hEPO levels were determined by ELISA asdescribed above. Red cell counts were done by hemocytometer, hematocritwas determined by centrifugation of blood in micro-capillary tubes, andhemoglobin concentration was analyzed by cyanmethemoglobin assay (DMA,Arlington, Tex.) according to manufacturer's specifications and comparedwith a standard (Stanbio Laboratory, San Antonio, Tex.) analyzed at 570nm on a spectrophotometer. Reticulocytes were analyzed by either newmethylene blue stain, or by FACS analysis of thiazole orange stainedperipheral blood samples (RETIC-COUNTO®, Becton-Dickinson, MountainView, Calif.); the results of data obtained by either of these methodswere similar.

An initial experiment revealed that high levels of hEPO and elevatedhematocrits were maintained for >100 days in mice injected IM with6.5×10¹¹ rAAV-hEPO. Next, adult female Balb/c mice were injected IM inboth hind limbs with a single administration of rAAV-hEPO at dosagesranging from 3×10⁹ to 3×10¹¹ particles. Control animals were injectedwith rAAV-LacZ. The resulting serum hEPO levels were analyzed and arereported below in Table II. As can be seen, a well-defined dose-responsewas obtained 20, 41, 62 and 83 days post injection.

The time course of hEPO secretion by animals receiving rAAV-hEPO isdepicted in FIG. 7. As can be seen, serum levels of hEPO increased withtime to plateau at from 6 to 8 weeks after injection.

The biological activity of secreted hEPO can be monitored by elevationof hematocrit in the experimental animals. A comparison of circulatinghEPO levels versus hematocrit is shown in Table II. The comparison showsthat hematocrit increased with time and increasing recombinant viriondose. Further, stable elevation in hematocrit has been observed for upto 40 weeks in a group of experimental animals injected with rAAV-hEPO.Control animals had undetectable levels of hEPO (<2.5 mU/mL, the lowerlimit of detection for the assay).

These results indicate that persistent and stable high-level secretionof hEPO, with a corresponding elevation in hematocrit, is establishedfollowing a single IM administration of-rAAV-hEPO.

In addition, comparison of the expression of hEPO by animals injected IMwith rAAV-hEPO (3×10¹¹ single-stranded genomes) and animals injected IMwith the pW1909EPO plasmid (1.4×10¹³ double-stranded genomes in 100 μgDNA) shows that the recombinant virions gave rise to significantlygreater levels of EPO expression. As reported in Table II, 20 dayspost-injection, recombinant virion-injected animals had serum levels of445±98 mU/mL, while the plasmid-injected animals had levels of 8 ±10mU/mL. At 41 days post-injection, levels in the recombinantvirion-treated animals had risen to 725±112 mU/mL, while the levels inthe plasmid-treated animals had dropped below the level of detection.The animals receiving rAAV-hEPO exhibited approximately 60-fold morecirculating hEPO with 100-fold less input genomes at 20 dayspost-injection, or approximately 6000-fold greater secretion per genome.At 41 days post-injection, this difference was even greater, since theplasmid expression was below the level of detection.

TABLE II EPO Expression and Hematocrit: rAAV-hEPO Dose-Response Daysafter Administration 20 days 41 days 62 days 83 days Dose EPO HCT EPOHCT EPO HCT EPO HCT 3 × 10¹¹ 445 ± 98  74.2 ± 1.2 725 ± 112 82.3 ± 1.2769 ± 61  86.5 ± 1.4 723 ± 253 88.5 ± 0.7 1 × 10¹¹ 85 ± 14 72.8 ± 1.5212 ± 23  79.5 ± 1.7 234 ± 75  83.2 ± 0.2 220 ± 51  83.2 ± 2   3 × 10¹⁰17 ± 5  60.0 ± 3.5 34 ± 17 74.7 ± 3.2 55 ± 28 78.7 ± 2.0 73 ± 45 80.0 ±3   1 × 10¹⁰ 3 ± 1 52.9 ± 1.8 11 ± 3  61.5 ± 1.9 12 ± 8  68.4 ± 4.6 15 ±5  70.8 ± 8   3 × 10⁹ <2.5 49.9 ± 1.4 <2.5 53.5 ± 2.5 <2.5 57.0 ± 2.4 4± 4 57.5 ± 3   i.v. 7 ± 3 54.7 ± 3.2  13 ± 2.0 <64.4 ± 5.3   10.1 ± 0.7 70.8 ± 8   21 ± 10 74.6 ± 7   Control <2.5 48.9 ± 1.0 <2.5 49.1 ± 0.8<2.5 48.1 ± 0.7 <2.5 48.2 ± 9   Plasmid  8 ± 10   50 ± 3.0 <2.5 50.2 ±1.0 <2.5 47.8 ± 0.9 N.D. N.D. Values representing means ± standarddeviation (SD). EPO = serum levels of human EPO (mU/mL) in Balb/c mice;HCT = hematocrit (%); N.D. = not done; i.v. = intravenous injection with3 × 10¹¹ rAAV-hEPO; Plasmid = injection with 100 μg plasmid DNA (1.4 ×10¹³ double-stranded plasmid molecules); Control = injection with 3 ×10¹¹ particles of rAAV-lacZ.

EXAMPLE 7 A Comparison of hEPO Secretion from rAAV-hEPO Administered byIM or IV Routes

A comparison of the circulating levels of hEPO resulting from IM and IVroutes of administration was analyzed to determine which method of genedelivery results in higher levels of systemic hEPO. Balb/c mice wereinjected with 3×10¹¹ rAAV-hEPO using either the IM route as describedabove, or intravenously (IV) in PBS in a total volume of 50 μL via thelateral tail vein. Serum hEPO levels were determined by ELISA using themethods described above.

As shown in Table II, hEPO levels resulting from the IV administrationswere significantly lower than the group that received the virions by theIM route. In particular, at 20 days post-injection, the IM routeresulted in levels of hEPO of 445±98 mU/mL, while the IV route-produced7±3.0 mU/mL. At 41 days post-injection, the EPO level observed with theIM route was 725±112 as compared with 13±2.0 mU/mL by IV, orapproximately 60-fold more efficacious. These data demonstrate that theIM route of injection resulted in higher systemic levels of hEPO, andsuggest that interstitial delivery in muscle results in improvedtransduction by the recombinant AAV virions.

EXAMPLE 8 In vitro and In vivo Transduction of Muscle Cells UsingrAAV-GAA Virions

Cardiomyopathy in infancy is frequently due to inherited metabolicdisease. One such metabolic disease, glycogen storage disease type II(Pompe's disease) is an inherited cardiomyopathy caused by a deficiencyin the lysosomal enzyme, acid α-glucosidase (GAA). GAA functions tocleave α-1,4 and α-1,6 linkages of lysosomal glycogen to releasemonosaccharides. Loss of enzyme activity results in accumulation oflysosomal glycogen in striated muscle, and is characterized by lysosomalrupture, contractile apparatus disruption and glycogen infiltration.Currently, no effective treatment is available.

Accordingly, the following studies were carried out to determine whetherthe recombinant AAV virions of the present invention can be used toobtain long-term expression of GAA in transduced muscle cells.

A. In vitro transduction of human skeletal muscle cells with rAAV-hGAAvirions.

Human skeletal myoblasts were seeded in cell culture plates at a densityof 2×10⁴ cells/cm², grown to confluence in appropriate growth media, andthen cultured for 14 days in human DM. Differentiation was verified bymicroscopic examination for multinucleate cells. In vitro transductionwas carried out by adding purified rAAV-hGAA virions at an MOI of 2×10⁵to the cultured myotubes in OptiMEM medium (Gibco BRL). DM was added tothe cultures after virion adsorption. Transduced control cultures wereestablished by transducing the myotubes with rAAV-LacZ at the same MOI,and negative controls were established by culturing non-transducedmyotubes.

Culture media was changed 24 hours prior to collection of supernatantsat day 3, 8 and 14 post transduction. hGAA expression was determined byenzymatic assay. Specifically, cell monolayers were harvested with0.050% trypsin in Puck's saline A containing 0.02%ethylenediaminetetraacetic acid. After quenching the trypsin with growthmedia, cells were centrifuged, the cell pellet washed with PBS, andresuspended in distilled water. Following three freeze-thaw cycles, thesamples were microfuged at 10,000×g. Protein in the supernatant wasdetermined using the Bicinchoninic acid method (Pierce) with bovineserum albumin (BSA) as the standard. GAA activity was measured usingcleavage of the glycogen analog 4-methylumbelliferyl-α-D-glucoside, by amodification of a previously described method (Galjaard et al. (1973)Clin. Chim. Acta. 49:361-375; Galjaard, H. (1973) Pediatr. Res. 7:56).Assays contained 30 μg protein in 125 μL water. Two volumes of 200 mMsodium acetate (pH 4.3) and 750 nM 4-methyumbelliferyl-α-D-glucoside(from a stock solution of 200 mM in dimethylsulfoxide) were added andthe samples incubated at 37° C. for one hour. Reactions were stoppedwith 625 μL sodium carbonate (pH 10.7). Once cleaved and alkalinized,the 4-methyumbelliferyl fluoresces. Measurements were made with afluorescence spectrophotometer with excitation at 365 nm and emission at448 nm. Assay measurements were compared against 4-methyumbelliferone(Sigma, St. Louis, Mo.) standards. Zero protein and zero time blankswere used.

The observed GAA activity is reported in FIG. 8 which demonstrates thatin vitro transduction of the human myotubes results in high level GAAexpression at day 8 and 14 post transduction.

B. In vivo transduction of skeletal muscle using rAAV-GAA virions.

Recombinant AAV virions encoding human GAA were administered to adultBalb/c mice in vivo to determine if systemic levels of GAA can beproduced by the transduced cells. Muscle tissue was isolated at varioustime points after administration, and processed for GAA activity usingan enzymatic assay.

In the study, tibialis anterior muscle in Balb/c mice was surgicallyexposed, and a single intramuscular injection into the left and rightmuscles (under direct vision) was used to administer the followingformulations: phosphate-buffered saline (PBS) alone (negative control);or PBS containing either 2×10¹⁰ rAAV-hGAA or rAAV-LacZ (for a total of4×10¹⁰ virions/animal).

Tissue samples of tibialis anterior muscle were obtained at 1, 4 and 10weeks following transduction. The tissue samples were prepared byhomogenization in water followed by three freeze-thaw cycles.Freeze-thaw lysates were microcentrifuged, and the resultant supernatantassayed for GAA activity as described above. The results of the studyare depicted in FIG. 9. As can be seen, stable expression of GAA in thetransduced mouse muscle cells was observed for ten weeks, demonstratingthat the recombinant AAV virions of the present invention are able toestablish efficient expression of a functional lysosomal protein intransduced cells, and thus provide a therapeutic approach for thetreatment of glycogen storage disease.

Accordingly, novel methods for transferring genes to muscle cells havebeen described. Although preferred embodiments of the subject inventionhave been described in some detail, it is understood that obviousvariations can be made without departing from the spirit and the scopeof the invention as defined by the appended claims.

Deposits of Strains Useful in Practicing the Invention

A deposit of biologically pure cultures of the following strain was madewith the American Type Culture Collection, 12301 Parklawn Drive,Rockville, Md., under the provisions of the Budapest Treaty. Theaccession number indicated was assigned after successful viabilitytesting, and the requisite fees were paid. Access to said cultures willbe available during pendency of the patent application to one determinedby the Commissioner to be entitled thereto under 37 CFR 1.14 and 35 USC122. All restriction on availability of said cultures to the public willbe irrevocably removed upon the granting of a patent based upon theapplication. Moreover, the designated deposits will be maintained for aperiod of thirty (30) years from the date of deposit, or for five (5)years after the last request for the deposit; or for the enforceablelife of the U.S. patent, whichever is longer. Should a culture becomenonviable or be inadvertently destroyed, or, in the case ofplasmid-containing strains, lose its plasmid, it will be replaced with aviable culture(s) of the same taxonomic description.

This deposit is provided merely as a convenience to those of skill inthe art, and is not an admission that a deposit is required. A licensemay be required to make, use, or sell the deposited materials, and nosuch license is hereby granted.

Strain Deposit Date ATCC No. pGN1909 Jul. 20, 1995 69871

                   #             SEQUENCE LISTING(1) GENERAL INFORMATION:    (iii) NUMBER OF SEQUENCES: 9(2) INFORMATION FOR SEQ ID NO: 1:      (i) SEQUENCE CHARACTERISTICS:          (A) LENGTH: 14 base  #pairs           (B) TYPE: nucleic acid          (C) STRANDEDNESS: single           (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #1:GGCAGCTGCC TGCA               #                   #                  #     14 (2) INFORMATION FOR SEQ ID NO: 2:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 14 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #2:GAAGGCGCGC CTTC               #                   #                  #     14 (2) INFORMATION FOR SEQ ID NO: 3:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 80 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #3:GCGGCCGCAC GCGTACGTAC CGGTTCGAAG CGCGCACGGC CGACCATGGT TA#ACTCCGGA     60 CACGTGCGGA CCGCGGCCGC             #                  #                   # 80 (2) INFORMATION FOR SEQ ID NO: 4:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 28 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #4:AAAATTCGAA CCTGGGGAGA AACCAGAG          #                  #             28 (2) INFORMATION FOR SEQ ID NO: 5:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 28 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #5:GTTTCCCCGC GAATGGACAA GCTTAAAA          #                  #             28 (2) INFORMATION FOR SEQ ID NO: 6:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 91 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #6:GCGGCCGCAC GCGTTGTTAA CAACCGGTTC GAAGCGCGCA GCGGCCGACC AT#GGGTTTAA     60 ACTCCGGACC ACGTGCGGAC CGAGCGGCCG C        #                   #          91 (2) INFORMATION FOR SEQ ID NO: 7:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 28 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #7:AAAATTCGAA CAGGTAAGCG CCCCTTTG          #                  #             28 (2) INFORMATION FOR SEQ ID NO: 8:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 28 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #8:GAGACCAAAG AGGGGTCCAA GCTTAAAA          #                  #             28 (2) INFORMATION FOR SEQ ID NO: 9:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 101 base #pairs           (B) TYPE: nucleic acid          (C) STRANDEDNESS: single           (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #9:GAGCTCAATC GATTGAATTC CCCGGGGATC CTCTAGAGTC GACCTGCAGC CA#CTGTGTTG     60 GATATCCAAC ACACTGGTAG GGATAACAGG GTAATCTCGA G    #                   #  101

What is claimed is:
 1. A method of delivering a selected gene to a heartof a mammalian subject, comprising: (a) providing recombinantadeno-associated virus (rAAV) virions which comprise an AAV vector, saidAAV vector comprising said selected gene, wherein said, selected geneencodes a therapeutic protein; (b) administering said rAAV virionsdirectly to said heart of said mammalian subject, wherein at least onecell of said heart is transduced by said rAAV virions; and (c)expressing said selected gene, wherein expression results in atherapeutic effect on a cardiomyopathy.
 2. The method of claim 1,wherein said therapeutic protein is missing or defective in said mammal.3. The method of claim 1, wherein said therapeutic protein is acidalpha-glucosidase.
 4. The method of claim 3, wherein said acidalpha-glucosidase is human acid alpha-glucosidase.
 5. The method ofclaim 1, wherein said cardiomyopathy is caused by an inherited disorder.6. The method of claim 5, wherein said inherited disorder is glycogenstorage disease type II.
 7. The method of claim 1, 5, or 6, wherein saidmammalian subject is a human.
 8. The method of claim 1, wherein saidadministering is done by injection.